Investigations were conducted to develop a general protocol for the isolation and in vitro culture of ovary‐derived cells from black crappie, Pomoxis nigromaculatus, and white crappie, P. annularis. Five digestive enzymes: 500 U/mL collagenase type I, 500 U/mL collagenase type IV, 0.05% trypsin‐EDTA, 0.25% trypsin‐EDTA, and 1X TrypLE™ Express were evaluated for live cell isolation. The isolated cells were cultured in 10 or 20% fetal bovine serum (FBS) in L‐15 growth media. In addition, four incubation temperatures (15°C, 20°C, 25°C, and 30°C) were also evaluated. The number of live cells obtained from the 0.25% trypsin and TrypLE™ Express treatments was significantly higher than other treatments. No difference in cell growth was observed between the two FBS treatments. Cells isolated using TrypLE™ Express and 0.25% trypsin reached 80% to 90% confluency in 12.5 cm2 cell culture flasks within 5 days of inoculation at 20 and 25°C. At 15°C, 10 days were required to reach 80%–90% confluency. Morphologically, cells incubated at 15°C, 20°C, and 25°C appeared healthier than cells incubated at 30°C, where irregular cell shape and substrate detachment were observed. We concluded TrypLE™ Express and 0.25% trypsin were optimal for cell dissociation and isolation of crappie ovarian cells. An incubation temperature of 20°C–25°C was favorable for cell culture in L‐15 media supplemented with either 10% or 20% FBS.
Every year globally 1.3 million people lose their lives from road traffic crashes (RTAs). Similarly, increasing rate of RTAs has been observed in Nepal including Kathmandu valley. This study is focused on the analysis of crash trends and respective site specific geometric features of urban road intersections in Kathmandu valley. Seventeen major intersections based on the data availability and traffic volume, are considered for the analysis of crash type. Previous crash data and traffic volume records of one year have been analysed. Common types of three and four legged intersections were taken for the study. Classified traffic volume at those intersections were converted into the Annual Average Daily Traffic. Evaluation factors for the crash analysis were determined by using predictive method. Crash frequency, crash rate, critical crash rate and crash prediction methods were used for ranking of the intersection. Priority for the safety improvement is prepared based on the results of this study.
Small recirculating aquaculture systems (RASs) are used to market live food fish, particularly in certain ethnic markets. We evaluated fish performance, water quality, and physiology of Channel Catfish (CCF) Ictalurus punctatus and Largemouth Bass (LMB) Micropterus salmoides that were harvested from ponds, transported, and held live at two stocking densities in a small RAS. One trial was conducted for each species, with two treatments (20 and 40 kg/m3) in each trial. Trials were conducted in individual RASs without feed during a holding period of 8 d. Water quality for each holding tank was measured at stocking and daily thereafter (24, 48, 72, 96, 120, 144, 168, and 192 h). Blood samples were obtained from fish to evaluate physiological response. Both CCF and LMB survived well in the system, with no mortality in either treatment. Average weight loss across both trials at a temperature of 15°C was 3.1% for CCF and 4.8% for LMB. Total ammonia nitrogen (TAN), un‐ionized ammonia (UIA), nitrite, and nitrate increased significantly over time in both trials. Mean water pH was significantly lower and mean TAN and UIA were significantly higher in the 40‐kg/m3 treatment for both trials. Blood glucose rose significantly after harvest and returned to a resting state during the holding period in both species. Blood sodium, potassium, and chloride also returned to a resting state within 24–72 h in the holding system. Estimated accumulation of TAN was 35.6 mg kg fish−1·d−1 for CCF and 21.6 mg kg fish−1·d−1 for LMB. Although the RAS biofilter was unable to convert ammonia to nitrate at the rate it was produced, the holding systems performed adequately at the densities evaluated. These trials provide data in support of the common practice of holding food‐size CCF and LMB at densities of 40 kg/m3 or less over a period of 8 d in a small RAS.
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